Effects of the bisquaternary ammonium compound hexaflourenium on the cholinergic system in the jejunum of the rat and the guinea-pig

EUROPEAN JOURNAL OF PHARMACOLOGY 15 (1971) 355-362. NORTH-HOLLAND PUBLISHING COMPANY

EFFECTS OF THE BISQUATERNARY HEXAFLUORENIUM

AMMONIUM COMPOUND

ON THE CHOLINERGIC

SYSTEM IN THE

JEJUNUM OF THE RAT AND THE GUINEA-PIG

A.H.J. SCAF Department of Pharmacology, State University, 1, Bloemsingel, Groningen, The Netherlands

Accepted 23 April 1971

Received 22 February 1971

A.H.J. SCAF, Effects of the bisquaternary ammonium compound hexafluorenium on the cholinergic system in the jejunum of the rat and the guinea-pig, European J. Pharmacol. 15 (1971) 355 -362.

1. The jejunum of the rat and the guinea-pig were used to study the effects of the bisquaternary ammonium compound hexafluorenium on muscarinic and nicotinic receptors, respectively. 2. Hexafluorenium 10-s M caused a contraction of the gut, which then became refractory to subsequent and similar doses, although the muscle continued to respond to nicotinic and musearinic drugs. 3. Hexafluorenium antagonized musearinic drugs competitively (PA2 = 5.7 ±0.2). The lack of strong muscarinic side-effects in clinical use, in spite of the inhibition of cholinesterase by hexafluorenium, could be explained by this antagonistic action. 4. The presence of hexafluoreni-, um (10--6-10-s M) increased the maximum response produced by nicotine. The log dose-response curve of nicotine is shifted to the right as a result of antagonism by hexafluorenium 10-s M. 5. Cholinesterase inhibition played a part in the effects of hexafluorenium on the gut. Hexafluorenium

Nicotinic receptor

1. INTRODUCTION The bisquaternary ammonium compound hexafluorenium bromide (hexamethylene-l,6-bis(9-fluorenyl-dimethyl-ammonium)dibromide or Mylaxen @) was synthesized by Cavallito et al. (1954). Macri (1954) described its neuromuscular blocking activity in laboratory animals. A preliminary clinical evaluation as a neuromuscular blocking agent is described by Cardaro and Arrowood (1955). These authors found that pretreatment with hexafluorenium greatly enhanced the neuromuscular blocking action of suecinylcholine. Rizzi and Galeotti (1956) observed that some side-effects o f the drug could be prevented by pretreatment with atropine. They remarked that cholinesterase inhibition could explain both these muscarinic side-effects and the potentiation of the suxamethonium block. Foldes et al. (1958, 1960) and

Muscarinicreceptor

Cholinesterase inhibition

Cavallito and Sandy (1959) showed that hexafluorenium inhibits true and pseudo-cholinesterase. Many authors have reported that in clinical use the muscarinic side-effects of hexafluorenium are surprisingly mild (Cordaro and Arrowood, 1955; Kok et al., 1962; McCaul and Robinson, 1962; Muteki et al., 1963; Jobidon et al., 1964; Cecconello et al., 1968; Figueora, 1968; Campbell and Swerdlow, 1969) in comparison with the effects of other cholinesterase inhibitors like neostigmine, physosti~aine and tetrahydroamino-acridine; doses of these drugs which potentiate the effect of suxamethonium produce strong muscarinic side-effects (McGaul and Robinson, 1962). The aim of this study was to discover why so few muscarinic side-effects of hexafluorenium are seen clinically in spite of its cholinesterase-inhibiting properties. For this purpose the influence o f hexafluoreni-

35 6

A.H.J.Sca~ Hexafluorenium and the cholinergic system

um on the nicotinic and muscarinic receptors in the gut was studied.

2. METHODS To study the effects on the muscarinic receptor the rat jejunum was used, because nicotine drugs have no effect on this tissue (Van Rossum, 1962a, 1963; Van Rossum and Van den Brink, 1963). Therefore, muscarinic effects of agonists that have both a muscarinic and a nicotinic activity can be determined. Acetylcholine, methacholine (acetyl-beta-methylcholine) and carbachol (carbamoylcholine) were used as agonists. For studying the effects on the nicotinic receptor the jejunum of the guinea-pig was used (Feldberg, 1951 ; Van Rossum, 1962a). Nicotine was chosen as the ganglion stimulating agent (Van Rossum, 1962a). Concentration-effect curves were constructed and evaluated. The log concentration-effect curves and the maximum effect of agonists were determined in the absence and presence of hexafluorenium. The maximum effect of a drug X (Exmax) is expressed as a proportion of the maximum effect that can be elicited (Emax) by a reference compound (methacholine). The negative logarithm of the concentration of the agonist X giving an effect of 50% o f E x max is used to indicate the position of the log concentration-effect curve (p[X] so)- The numerical values of these parameters are equal to those for the intrinsic activity and the affinity according to Ariens (1954) and Ariens and Van Rossum (1957). These symbols were chosen so that similar symbols could be used in the absence and the presence of other drugs. In the presence of other drugs the symbols E~ max and p[X] so are used. The symbol pA2 is used as defined by Schild (1947, 1949). For muscarinic drugs cunm!ative concentrationresponse curves can be constructed (Van Rossum, 1963; Van Rossum and Van den Brink, 1963). To obtain these curves the following procedure was adopted; first, Ema x was determined. For this purpose concentration-response curves of the reference compound methacholine were made until the height of two successive curves differed less than 10%. The maximum height of the last curve was considered to be the Ema x. Two concentration-effect curves of the

muscarinic agonist under consideration were then obtained; one without hexafluorenium and one after pre-incubation with hexafluorenium for a period of 1 min. To evaluate the influence of the inhibition of cholinesterase by hexafluorenium, neostigmine was used in some experiments. In these instances three curves were compared, one blank and the other two after incubation for 1 min with neostigmine and neostigmine plus hexafluorenium respectively. These experiments were compared with experiments in which only the agonist was used. With nicotinic drugs, concentration-effect curves can only be obtained by determining the responses to individual concentrations of the drug (Van Rossum, 1962a). In this study a geometrical sequence of concentrations of the nicotinic agonist was used with a ratio of ~/10. When the response to a dose of agonist had been determined, this dose was removed by changing the solution in the muscle-bath three times. Every 3 rain one point of the curve was determined. Between two curves, the waiting time was 15 min. The maximum contraction evoked with a muscarinic reference compound (methacholine) was considered to be also Ema x for nicotinic drugs (Van Rossum, 1962a). The rest of the procedure was the same as described for the cumulative experiments. The number of experiments with each muscarinic drug was 10, with nicotine6. A dose of either hexafluorenium or neostigmine or a dose of the combination was tested only once with the gut of one animal, so that all observations with one dose were independent in this respect. The laboratory" animals were: 1. Female Wistar rats (180-200 g), bred in our laboratory. 2. Female guinea-pigs ( 3 0 0 - 5 0 0 g ) , supplied by TNO, Zeist, the Netherlands. The Krebs solution contained in g/l: Na ÷, 3.27, K ÷, 0.23, Ca 2÷, 10, Mg 2+, 0.029, C1-, 4.45, HCO3-, 1.385, SO4 2-, 0.114, PO43-, 0.114 and glucose, 3.4. It was gassed with 95% 02 and 5% CO2. Contractions were recorded with the following system: 1. A Hottinger-Baldwin isotonic displacement transducer No. W 10. The tension applied to the muscle was 0.8 g. 2. A Hottinger-Baldwin measuring bridge No. KW 31II-5. 3. A Varian recorder No. G-14.

A.H.J.Sca£ Hexafluorenium and the cholinergic system

The following drugs were used: acetylcholine iodide, acetyl-beta-methylcholine chloride and carbamoylcholine chloride (Fluka), hexafluorenium bromide (Neissler, Mallinckrodt), neostigmine methylsulphate (E. Merck) and nicotine hydrogen tartrate

(B.D.H.). 3. RESULTS 3.1. The interaction between hexafluorenium and muscarinic drugs The smooth muscle cells in the gut not only contain muscarinic receptors, but are also associated with the enzyme acetylcholinesterase. If a drug acts on muscarinic receptors and is also susceptible to acetylcholinesterase, both these factors will influence the shape of the dose-response curve. Of the muscarinic agonists, which were used in this study, acetylcholine is broken down faster by acetylcholinesterase than methacholine, while carbachol is not broken down at all (Koelle, 1965). Fig. 1 shows that in rat jejunum the log d o s e response curves of the agonists are shifted to the right by hexafluorenium and the parallelism of the curves suggests that the latter may be a competitive antag-

35 7

onist of muscarinic drugs. However, in half of the experiments Ema x could not be obtained in the presence of hexafluorenium (fig. 2); in these cases the dose-response curve broke off sharply and, furthermore, the muscle was unable to sustain the contraction induced by the muscarinic agonist. The interaction still appeared to be of a competitive type as shown by a Lineweaver-Burk plot of the results obtained with carbachol (fig. 3), where the lines intersect near the y-axis. The affinity of competitive antagonists for their receptors can be expressed by means of pA2-values (Schild, 1947, 1949) and that of hexafluorenium for the muscarinic receptor is 5.7 -+ 0.2 (fig. 4). The effect caused by hexafluorenium 120 × 10-6 M disappeared 30 min after washing the preparation every 3 min with hexafluorenium-free Krebs, which demonstrated that the binding of the drug to these muscarinic receptors was reversible. Fig. 1 also shows that the same concentration of hexafluorenium did not shift the log dose-response curves of the different agonists to the same degree. Thus, it produced the smallest effect on acetylcholine and the largest on carbachol, a difference which might be expected if cholinesterase inhibition plays a part in the action of hexafluorenium on the gut. That the shift was determined by two processes is shown in

% contraction rat jejunum + Hexafluorenium 100 x I136 M.

i

I i

eO

20

40

20

-8 10

-7 10

a. Acetylcholine

106M.

-8 10

'-7 10

b. Methacholine

'.6 10 M.

-8 10

-7 10

_6 10 M.

c. CarbachoL

Fig. 1. T h e interaction o f h e x a f l u o r e n i u m with muscarinic drugs. Log d o s e - r e s p o n s e curves representing the m e a n results of 10 experiments. T h e m e a n s were d e t e r m i n e d according to the following procedure. Of all 10 curves the logarithms of the concentrations giving 10, 20, 40, 50, 60, 80 and 90% o f E m a x were determined by interpolation from the log d o s e - r e s p o n s e curve. T h e m e a n s o f these values are presented here. Only those parts o f the curves are shown that occurred in all 10 individual curves. T h e influences of 1.2 X 10"~. 12 X 10-6 and 120 X 10--6 M h e x a f l u o r e n i u m on the curves of all these agonists have been studied, b u t only those curves that can be distinguished from the blank ones are shown.

A.H.J.Scaf, Hexafluorenium and the cholinergic system

358

Emox

6

E+0.05Emo x -6 +Hexofl.uorenium xl0 N.

7

,j

2

1 (a)

3

4

16 7

% controction rot jejunum + HexofLuorenium 100

80. 6O

[ Corbochol. ] Fig. 3. The interaction between hexafluorenium and carbachol: Lineweaver-Burk plot. In pharmacological experiments, the effect values need to be corrected to allow for receptor occupancy at threshold (Ariens, 1964, p. 403). In these experiments the factor was 0.05 Ema x. The data were the same as those in fig. lc. The intersection of the lines near the y-axis strongly suggests competitive antagonism.

4O

[[Carbachot]50' /

2O

Log l - - ~ ~ -1 ~[Corbecho!.l 50 2

'-8 10

-7 10 (b)

' 106M.

Fig. 2. An experiment with 1.2 X 10-s M hexafluorenium and methaeholine, a) A sample o f the tracing obtained. The two curves on the left are controls; the hexafluorenium was added at the arrow. Note that the highest concentration o f methacholine used in the presence o f the hexafluorenium caused a short lasting contraction. The numbers indicate the negative logarithm of the concentrations o f agonist, b) Log dose-response curves constructed from the data shown in fig. 2a. Note that the curve obtained in the presence o f hexafluorenium is parallel to that of the controls, although Ema x could not be obtained.

Fig. 4. The interaction between hexafluorenium and carbachol: Arunlakshana-Schild plot. The data o f fig. lc have been used. The pA 2 is 5.7 -+ 0.2.

fig. 5, where the maximum difference in displacement of the curves of the agonists was reached at a concentration of 12 × 10-6M hexafluorenium; a further increase to 120 X 10-e M did not cause any additional increase of this difference. If this was

caused by cholinesterase activity in the gut, then it should be reduced if the enzyme was already inhibited by a second inhibitor; for this purpose neostigmine was used. The shift of the log dose-response curves of the agonists caused by neostigrnine was first

0

J 1'.2

1'2

iio 166M.

Hexefl.uorenium

A.H.ZScaf, Hexafluorenium and the cholinergic system

359

determined with a concentration of neostigmine one third of that required to produce a contraction (table 1). The dose-response curves of the agonists in the presence of the same concentration of neostigmine and 12 X lO-6M hexafluorenium were then obtained. Fig. 5 shows that under those conditions the difference in shift between carbachol and methacholine disappeared while that between carbachol and acetylcholine was almost halved.

P[X]~o-P[X]50 0.0.

-0.2. 0./.,

0.6. 0.8,

3.2. The interaction between hexafluorenium and

- 1.0,

nicotine -1.2. -1.3.

()

112

1'2

1½0

x 1()6H. H e x a f t u o r e n i u m

Fig. 5. The effect of increasing concentrations of hexafluorenium on the position of the log dose-response curves of acetylcholine (zx), methacholine (o) and carbachol (D). The open symbols indicate the shift of the curves (expressed as p[X]~o - p[X]s o) of the different agonists caused by hexafluorenium, while the closed symbols represent the shift of the curves caused by hexafluorenium in the presence of neostigrnine as described in table 1. Note: 1. The curves of the three agonists were not shifted to the same degree by hexafluorenium. The maximum difference is reached at 12 × 10-6 M hexafluorenium, as can be seen from the parallel course of the lines above this concentration. 2. In the presence of neostigmine the difference in shift between methacholine and carbachol has disappeared and between acetylcholine and carbachol has almost been halved. Table 1 The shift of the log dose-response curves of the muscarinic drugs caused by neostigmine*. p[X] ~o-pIX] so ± S.E.M. Acetylcholine Methacholine Carbachol

0.30 ± 0.03 0.22 ± 0.04 0.02 ± 0.03

* The concentration of neostigmine was one-third of the concentration causing a contraction. The concentration that had to be used was determined for each piece of jejunum separately and was 10-s,8±°,1 M. A positive value in the table corresponds to potentation. The number of experiments was 10.

In experiments with rat jejunum, it was found that cholinesterase inhibition partly explained the effects of hexafluorenium upon the smooth muscle. In experiments with guinea-pig jejunum, the combination of hexafluorenium and neostigmine was again used for the same reasons as those given in section 1. In preliminary experiments with guinea-pig jejunum, concentrations of neostigmine higher than 10-8 M sometimes produced a contraction, so that a concentration of 10-SM was used throughout all subsequent experiments; the results of these experiments are summarized in table 2. Both hexafluorenium and neostigmine increased the maximum contraction which could be obtained with nicotine. Hexafluorenium 10-s M shifted the log dose-response curve to the right, thus indicating an antagonistic effect. The same displacement was seen whether neostigmine was present or not. A shift to the left was obtained with the combination of neostigmine with hexafluorenium 10-~ M while neostigmine alone caused no shift at all. In 10 out of 36 experiments, the first dose of nicotine caused a large contraction in the presence of hexafluorenium, although the concentration of nicotine was as low as 3 X 10-7 M. A subsequent injection of the same dose of nicotine produced no contraction, but a dose-response curve of nicotine could be obtained using higher concentrations of the drug. 3.3. The stimulant action o f hexafluorenium In addition to those effects already described, concentrations of hexafluorenium higher than 3 × 10-6 M produced a contraction of the gut, up to 50% of the maximum contraction that could be obtained with methacholine. The preparation relaxed 2 min

A.H.J.Scaf, Hexafluorenium and the cholinergic system

360

Table 2 The action of hexafluorenium on the effects of nicotine. The number of experiments was 6. 1

2

3

4

5

6

7

8

9

[Hfl]

Nst

EN max

EjV max

4-3

Significant

p[N] so

p[N] ~0

8-7

0 0 10- 7 10-7 10-6 10 -6 10-5 10-s

+ + + +

0.62 0.68 0.72 0.69 0.57 0.73 0.71 0.59

0.63 0.81 0.80 0.84 0.74 0.93 0.93 0.99

0.01 0.13 0.08 0.15 0.17 0.19 0.22 0.40

+ 4~ + + + +

5.17 5.30 5.33 5.23 5.40 5.28 5.42 5.35

5.13 5.33 5.32 5.26 5.47 5.42 5.19 5.13

-0.03 0.02 -0.02 0.03 0.07 0.13 -0.23 -0.22

10 Significant + + +

1 Molar concentration of hexafluorenium. 2 Absence or presence of neostigmine, 10 -8 M. 3 Maximum effect of nicotine as a fraction of Emax determined with methacholine (S.E.M.: 0.04-0:07). 4 Maximum effect of nicotine as a fraction of Ema x after pre-incubation with the concentrations of hexafluorenium and neostigmine indicated in columns 1 and 2 (S.E.M.: 0.02-0.08). 5 Mean of the differences of E}v max and EN max: A positive value indicates potentiation (S.E.M.: 0.02-0.10). 6 + indicates that the difference between 4 and 3 is significant (P = 0.05) with the Wilcoxon matched-pairs signed-ranks test (Blalock, 1960). 7 Negative logarithm of the concentration of nicotine giving an effect of 50% of E N max (S.E.M.: 0.05-0.15). 8 Negative logarithm of the concentration of nicotine giving an effect of 50% of E ~ max after pre-incubation with the concentrations of hexafluorenium and neostigmine indicated in columns 1 and 2 (S.E.M.: 0.07-0.15). 9 Mean of the differences of p[N] ~o and p[N] 5o. A positive value indicates potentiation (S.E.M.: 0.02-0.10). 10 + indicates that the difference between 8 and 7 is significant (P = 0.05) with the Wilcoxon matched-pairs signed-ranks test (Blalock, 1960).

after w a s h i n g o u t t h e drug, b u t a s u b s e q u e n t dose o f h e x a f l u o r e n i u m was always i n e f f e c t i v e . O n t h e o t h e r h a n d , a n a d e q u a t e dose o f a n i c o t i n i c or m u s c a r i n i c agonist still i n d u c e d a c o n t r a c t i o n , as d e s c r i b e d in t h e s e c t i o n s 1 a n d 2. F u r t h e r m o r e , in f o u r e x p e r i m e n t s w i t h t h e j e j u n u m o f t h e rat this e f f e c t o f h e x a f l u o r e n i u m c o u l d n o t be p r e v e n t e d b y p r e t r e a t m e n t w i t h a t r o p i n e 10 -6 M, t h e pA2 o f a t r o p i n e b e i n g a b o u t 9.

f l u o r e n i u m a n d c a r b a c h o l is c o m p e t i t i v e on t h e m u s c a r i n i c r e c e p t o r , t h e pA2 b e i n g 5.7 --- 0.2 (figs. 1, 3 a n d 4). This a n t a g o n i s m was p a r t l y m a s k e d in t h e cases o f a c e t y l c h o l i n e a n d m e t h a c h o l i n e (figs. 1 a n d 5), w h i c h c a n be e x p l a i n e d b y t h e c h o l i n e s t e r a s e inhibiting property of hexafluorenium. An indication t h a t c h o l i n e s t e r a s e i n h i b i t i o n m a y play a role is also f o u n d in t h e i n f l u e n c e o f n e o s t i g m i n e o n this phen o m e n o n . W h e n this d r u g was p r e s e n t t h e curves o f a c e t y l c h o l i n e a n d m e t h a c h o l i n e were s h i f t e d m o r e in the directions of antagonisms by hexafluorenium

4. D I S C U S S I O N T h e d r u g p a r a m e t e r s o f t h e agonists f o u n d in these e x p e r i m e n t s are c o m p a r a b l e w i t h t h o s e given b y o t h e r s ( V a n R o s s u m , 1 9 6 2 a , 1 9 6 3 ; ,Ariens, 1 9 6 4 ; Bosse a n d W a s s e r m a n n , 1 9 6 7 ) . T h e affinities are given in t a b l e 3. T h e E m a x o f n i c o t i n e is 0 . 6 8 + 0.03 (n = 16), while a c e t y l c h o l i n e a n d c a r b a c h o l h a v e t h e same E m a x as m e t h a c h o l i n e . T h e results o f t h e s e m i l o g a r i t h m i c a n d t h e L i n e w e a v e r - B u r k p l o t s indicate t h a t t h e a n t a g o n i s t i c i n t e r a c t i o n b e t w e e n h e x a -

Table 3 The affinities of acetylcholine, carbachol, metacholine and nicotine.

Acetylcholine Carbachol Methacholine Nicotine

p[X] so + S.E.M.

n

7.11 7.46 7.34 5.31

10 10 10 16

± 0.06 ± 0.05 ± 0.02 ± 0.06

A.H.J.Scaf, Hexafluorenium and the cholinergic system

than when it was absent (fig. 5). Although no indications were found for a non- or un-competitive interaction between hexafluorenium and the muscarinic drugs in figs. 1 and 3, Ema x of these muscarinic drugs could not be obtained in 50% of the experiments with hexafluorenium. This strange type of antagonism cannot be explained by any current theory of pharmacological action. The interaction between hexafluorenium and nicotine was also complex. The actions of hexafluorenium 10-6 M and of neostigrnine 10-8 M on the effects of nicotine were very much alike. Both drugs increased EN max without an alteration of the p[N] so. This can be explained by the inhibition of the acetylcholinesterase by these drugs. Assuming that both the relation between the concentration of nicotine and the number of nicotinic receptors occupied in the ganglion and the relation between this degree of occupation and the amount of released acetylcholine in the terminal synapse are unaltered, this amount of transmitter can evoke an increased response in the muscle if it is not broken down during diffusion to the muscarinic receptors. From the experiments with rat jejunum it was concluded that hexafluorenium has atropine-like properties. Since atropine-like drugs behave as non-competitive antagonists of nicotine (Van Rossum, 1962b), it is possible that the log d o s e response curve of nicotine is altered by hexafluorenium in the direction of potentiation, by inhibition of cholinesterase and in the direction of antagonism by blockade of the muscarinic receptors. Therefore, in these experiments, the potentiation would be less than might be expected after inhibition of cholinesterase alone. Cholinesterase inhibition may also be the cause of the small shift to the left found with the combination of hexafluorenium 10-6 M and neostigmine 10-8 M. Such a displacement may be expected when an incomplete occupation of the nicotinic receptors causes a release of acetylcholine great enough to evoke the maximum effect of acetylcholine, if it is not broken down by cholinesterase. Beside these effects, competitive antagonism between hexafluorenium and nicotine may exist at a concentration 10-s M hexafluorenium. This antagonism only exists at a concentration higher than the muscarineblocking and cholinesterase-inhibiting concentrations. Hexafluorenium itself stimulated the gut, but how it does so is not clear. The cholinergic system is

361

unlikely to be involved, for when hexafluorenium stimulated the muscle via this system, the contraction would be prevented by atropine 10-6 M, this concentration being 1000 times the concentration that corresponds to the pA 2 value. Combining the results collected with the jejenum of rat and guinea-pig, it is difficult to predict what actions might be expected in vivo. The pharmacological literature gives little information about the effects of hexafluorenium on the autonomic nervous system. Della Bella and coworkers (1961, 1962)have described a blockade of the effects of electrical preganglionic stimulation of the nerve in the isolated vagus-heart preparation of the guinea-pig and the isolated vagus-stomach preparation of the rat by hexafluorenium in a concentration of about 10-s M. They ascribed this to a ganglionic blocking activity because they assumed that bisquaternary ammonium compounds do not possess atropine-like properties. On the other hand, no ganglion blocking effects have been found in rabbits (Della Bella et al., 1961) or dogs (Macri, 1954) so that the parasympatholytic activity described in the present study is a more likely explanantion for the block found by Della Bella et al. (1961, 1962) than blockade of the ganglion. In the clinical literature most authors agree that hexafluorenium has only weak muscarinic side-effects in spite of the inhibition of cholinesterase by this drug. This lack of severe side-effects may also be attributed to the atropine-like properties of hexafluorenium.

ACKNOWLEDGEMENTS I am indebted to MaUinckrodt Chemical Works (St. Louis, U.S.A.) for supplying hexafluorenium bromide (Mylaxen ®) through the mediation of Nourypharma (Oss, The Netherlands). I thank Mr. R. Makken for technical assistance and Prof. J.W. Thompson, New Castle, for his valuable criticism.

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362

A.H.J.Scaf, Hexafluorenium and the cholinergic system

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